The anticodon stem loop (ASL) is critical for decoding properties of tRNA. The universal threonylcarbamoyladenosine (t6A) and the widespread 7-deazaguanosine derivative queuosine (Q) are two ASL modifications at positions 37 and 34, respectively. Our discovery of the biosynthesis pathways for these two complex modifications that had been missing for decades has opened the path to study the role of these modifications in vivo. The long-term goal of our research is to expand fundamental knowledge on the synthesis and function of complex tRNA modifications and related molecules, and to understand their roles in core cellular processes and physiology. The current application focuses on the in vivo role of t6A and Q. Because their pathways are complex and draw on primary metabolites and both these modifications have central roles in decoding, t6A and Q are ideal candidates for molecules that integrate metabolism and translation and could play unforeseen regulatory roles. Preliminary results suggest that the absence of t6A triggers an unfolded protein response in both yeast and Bacteria and that t6A affects the translation of specific proteins and this will be the focus of Aim 1. Preliminary phenotypic screens show that Escherichia coli mutants that lack Q have metal sensitivity or resistance phenotypes, and the role of this modification in how cells sense and adapt to metal stresses will be explored in Aim 2. In addition, it has recently been shown that RNA and DNA modifications pathways have much more in common than previously anticipated. Our unexpected discovery that Q precursors are inserted in DNA suggests that paralogs of Q synthesis enzymes have been recruited in DNA metabolism and this will be studied in Aim 3. Aim 4 will focus on the potential role in DNA repair of a paralog of the first enzyme of t6A synthesis. The approach is innovative because comparative genomic methods were used to guide the experimental effort, and this work is revealing new regulatory mechanisms linking metabolism and translation as well totally novel modifications of DNA. The proposed research is significant because it will advance our understanding of the role of critical tRNA modifications and novel DNA modifications in fundamental cellular physiology.